JPH087186B2 - Thermal technique of bulk fluid motion in capillary electrophoresis. - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to capillary electrophoresis, and more particularly to a method of moving a fluid in a fast bulk flow to or through a capillary electrophoresis column with a primary focus on sample loading.

BACKGROUND AND SUMMARY OF THE INVENTION Capillary electrophoresis is a technique of considerable interest for the analysis of biological mixtures, especially small peptide, protein and nucleic acid mixtures. Because it is used with very few samples and allows the use of high voltages, thereby achieving separation at high speeds.

One of the problems in capillary electrophoresis is its placement inside the ends of the capillaries in preparation for sample loading or separation. Currently, this is usually accomplished by electrophoretic, electroosmotic or differential pressure techniques.

In electrophoretic packing, a high voltage is used for a short period of time to transfer the sample from the sample reservoir to the capillary. The purpose is to move all species in a small sample into the capillary a short distance. Once this has been done, the sample reservoir is replaced with an appropriate buffer solution,
It will be possible to perform a packed type of electrophoretic separation.

What really happens with electroosmotic packing is a combination of electrophoresis and electroosmosis, with electroosmosis having the major effect. However, electroosmosis varies from experiment to experiment, creating difficulties in reproducibility of sample volume.

Electroosmosis is suppressed by the application of a suitable coating on the inside of the capillaries, leaving electrophoresis as the only driving force for sample injection. However, electrophoresis has its own disadvantages. These result from the differences that are necessarily present in the various species in the sample due to their response to electrical potential. These differences affect the distance that the species travel into the capillary during filling and thus the amount of each species entering the capillary. The slower-migrating substance will therefore migrate a shorter distance into the tube than the faster-migrating substance. Therefore, depending on the filling conditions, the composition of the applied sample is
It may differ from that of the original sample.

Other variations in electrophoresis, such as variations in the strength of the current, must also be taken into account. The magnitude and importance of these changes can vary.

In hydraulic filling, sample introduction is achieved by a pressure differential between the capillaries, either by applying a partial vacuum at the outlet or by applying a positive pressure at the inlet. The problem of hydraulic filling arises from the limited degree of control over the pressure differential and its duration (two important parameters that together govern the volume of sample introduced).

Capillary of an electrophoresis system to a very precise and reproducible extent by imposing a controlled temperature change on the contents of either the tube or a closed container in liquid communication with the tube. It has been found that can be achieved in. This finding is applicable to the introduction of small volumes into a tube for purposes such as sample loading as well as the transfer of large volumes to or through the tube for purposes such as flushing the tube with buffer.

The change in temperature is a decrease or increase in temperature and occurs between all initial and final temperatures resulting in controlled thermal contraction or expansion. Temperatures that are not involved in the phase change of the heated or cooled medium and thus result in a continuous volume change would be preferred. If a temperature change is applied to the capillary itself, the temperature is most conveniently selected and the final temperature is the temperature at which the separation should take place.

Thus, one aspect of the invention involves imposing a temperature change on the capillary itself. These embodiments are extremely useful for loading samples into capillaries. In these embodiments, the capillaries are filled with a separation medium, and the temperature of the filled capillaries is adjusted as necessary to provide a temperature drop of a magnitude calculated to achieve a selected volume of contraction. . The magnitude of the temperature drop is of course determined in relation to the length of the capillary to which the temperature drop is applied, and this length is
Either the whole capillary or a selected portion of its length.

Once the capillary temperature equilibrates to the initial temperature, the end of the capillary into which the sample is to be introduced is submerged in the reservoir containing the sample. The temperature of the capillary or its temperature controlling part is then reduced to the final temperature, drawing a portion of the sample into the end of the capillary. The final temperature is maintained when the reservoir is then replaced by the electrode buffer and electrophoresis is performed. Appropriate and accurate selection of both the length of the capillary undergoing the temperature change and the magnitude of the temperature change will result in an accurate and very accurate determination of the size of the sample without changing the proportion of the sample components as they enter the capillary. Allow reproducible control.

In another aspect of the invention, the temperature change is made to a sphere or other external container that is coupled to one end of the capillary which is filled with liquid and is in complete liquid communication with the contents of the capillary. This causes bulk fluid motion without any temperature change in the capillary itself. Depending on its position, the sphere pushes or pulls liquid through the capillary and does it directly or through an intervening reservoir. The change in temperature is therefore either rising or falling. The use of spheres provides a wide range of volumetric flow, as it is not limited to capillary size. When the volume of the sphere is large compared to the volume of the capillary, filling of the sample can be achieved with very small changes in temperature. However, the spheres are particularly useful for flushing whole capillaries with buffer.

Bulk fluid motion by either of these methods, whether it is sample loading or a full capillary flush, can be performed with the same accuracy as a thermometer. Other benefits, features and aspects of the present invention will be apparent from the description below.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a diagram of the general approach to the present invention where changes in temperature are applied to the capillary itself.

FIG. 2 is an explanatory view of a special mode of the present invention, which is partially cut open and which utilizes the principle of FIG.

FIG. 3 is a diagram of a system according to the invention in which a temperature change is applied to a sphere external to a separate part of the capillary.

FIG. 4 is a diagram of an automated system according to the present invention that also utilizes temperature controlled spheres.

Detailed Description and Preferred Embodiments of the Present Invention The present invention can work with a wide variety of electrophoretic separation media. Placed inside the capillary, and depending on the more specific aspect,
Any medium that can contract or expand due to changes in temperature or that is drawn a distance through the tube by a medium that contracts or expands within the container that is coupled to the tube is used. Possibility is
Both gels and solutions, which are generally aqueous solutions, include solutions of highly viscous polymer solutions and simple buffer solutions. Aqueous buffer solutions are preferred.

The description below begins with the manner in which the temperature change is imposed on the capillary itself, followed by the manner in which the temperature change is imposed on the sphere outside the capillary but in fluid communication with the interior of the capillary.

In embodiments in which the temperature of the capillaries varies, the size of the capillaries, both diameter and length, can be varied widely, without any rigor. Often, capillaries with internal diameters of about 10 to about 200 microns are used, preferably about 20 to about 100 microns. All capillaries are temperature controlled to achieve migration. However, in many cases, it would be more convenient to impose temperature changes on only a portion of the capillary due to the need for a liquid tight connection at the ends of the capillary. In capillaries where on-line detection is used, the point of detection is either at or below the temperature control portion of the capillary.

The length of the temperature control portion can vary widely depending on the size of the portion sample desired and the preferred range of temperature differences and the coefficient of thermal expansion of the separation medium. In many cases,
Best results are from about 50 to about 2000 mm, preferably about 100 to about 100.
It will be achieved with a temperature control length of 0 mm.

A wide range of volume samples can be filled by this approach. Many separations to which the present invention applies are approximately 0.0003 by volume.
~ About 0.03 µl, and preferably about 0.0005 to about 0.005 µl, is contemplated to contain the sample. For capillaries with an inner diameter of 0.05 mm, partial samples within these ranges range from about 0.15 to
It will extend into the capillary for a distance of about 15 mm, preferably 0.25 to about 2.5 mm.

The required temperature change is easily calculated from the length of the temperature control section, the size of the partial sample and the coefficient of thermal expansion of the separation medium. In many cases, a temperature difference of at least 5 ° C, preferably at least 10 ° C, more preferably about 10 to about 70 ° C, and more preferably about 20 to about 40 ° C will be used.

Preferred methods of carrying out the invention according to these aspects are:
The pulling of the separation medium inwardly from only one end of the capillary by the drop in temperature. In this way, a portion of the sample will fill the difference in total volume resulting from the contraction of the separation medium. This makes the calculations that determine the temperature and increases the control of the sample volume. This can be accomplished by any technique readily apparent to one of ordinary skill in the art for impeding or blocking the flow of liquid at the opposite end of the capillary. One of the techniques is to submerge the opposite end in a sample solution, preferably a liquid having a higher viscosity than both the sample solution and the separation medium, thereby providing greater resistance to flow at that end than at the end of sample loading. Is. Solutions of polymers such as methylcellulose will work effectively as high viscosity liquids. On the other hand, the end of the tube could be closed by a dialysis membrane or gel plug. The reduction in temperature can be achieved by any conventional means, and various possibilities will be readily apparent to those skilled in the art. Submerging the capillary in, or thereby washing, its exterior in a suitable temperature of cooling liquid is the most convenient way to achieve this result. This always brings other advantages of temperature control in the capillaries. Precise control of temperature can significantly increase the reproducibility of the experiment, noting that the electrophoretic velocity can change about 2.5% with every 1 ° C change in temperature. Continuous cooling of the capillaries is also one of the most attractive features of capillary electrophoresis, which allows the use of high voltages, which can shorten the time of the experiment.

Once a portion of the sample has been loaded onto the capillary column,
Remove the sample reservoir and replace with an appropriate buffer solution for electrophoresis. Care must be taken to prevent loss of the partial sample from the capillary during this exchange. This can be achieved by keeping the temperature of the capillaries at the temperature used for filling so that the separation medium does not expand or contract. The components of the system can also be designed with appropriate channel and sphere arrangements between different fluid reservoirs so that switching can be done without disconnecting any parts.

Referring to FIG. 1, a somewhat basic system sketch is shown to illustrate the general concept of the invention.
A capillary of fused silica 11 is wrapped around a support rod 12 inside a tray 13 filled with a temperature controlled heat transfer liquid. The two ends 14, 15 of the capillaries extend outside the tray 13 into the external reservoirs 16, 17.

For sample loading, the capillary 11 is filled over its entire length with a separation medium, the outlet reservoir 17 is filled with a buffer solution, preferably one containing a viscosity-increasing solute, such as methylcellulose, and the inlet reservoir 16 is the sample. Filled with solution. The exposed ends 18, 19 of the capillaries are submerged in the corresponding solution. Heat transfer medium in tray and 2
All media, including the solution in each of the individual reservoirs, are at the starting temperature. With the media in place in this way, the filling of the sample is achieved by replacing the heat transfer liquid in the tray with a cooler heat transfer medium, the temperature difference being the length of the capillaries in the tray, the diameter of the capillaries and Selected based on the coefficient of thermal expansion of the separation medium in the capillary to achieve the desired volumetric shrinkage of the separation medium and draw the same volume of sample from the reservoir on the inlet side.

Once the system has equilibrated, the sample solution in the inlet reservoir is replaced with buffer and the electrophoresis proceeds with the cold heat transfer medium retained inside tray 13. Detection in this embodiment is accomplished by the ultraviolet beam 20 passing through the capillary at a point outside the cooling tray 13 opposite the sample injection end. Other conventional methods of detection such as fluorescence can also be used.

The embodiment shown in FIG. 2 accommodates a capillary 31 and acts as a jacket or enclosure for the passage of heat transfer fluid.
Including 30. The inlets and outlets 32, 33 for the heat transfer fluid are
A fluid is circulated continuously around the outside of the capillary. The internal baffle 34 guides the flow of fluid. The ends of the capillaries are sealed to conical inlets and outlets 35, 36, which connect the interior of the capillaries to the exterior of the cartridge. These conical ports coincide with similarly shaped joints 37, 38, 39 in the fluid reservoir block due to the pressure seal when the parts are assembled in a suitable support block.

The sample solution and the buffer solution displacing the sample solution are either in different blocks 40, 41 as shown or in separate compartments with joints controlled by spheres that are manually or automatically operated. Combined in one block. On the other hand, the selection between the sample solution and the electrode buffer solution can be controlled by a flash mechanism that is designed to be generated at regular time intervals.
The outlet block 42 contains the buffer solution used at the capillary outlet. Electrode junctions 43, 44 are shown in each block. Detection is accomplished by an ultraviolet beam passing through a window 46 aligned with the capillary at a point near the outlet 38.

A generalized example of these embodiments in which a temperature difference is applied below the capillary sphere is shown in FIG. Separation of the electrophoretic sample in this arrangement occurs at the portion of the capillary 51 shown to the right of the sphere 52. The sample is introduced at the right end 53 of the capillary and the sample components migrate to the left during electrophoresis in the direction indicated by arrow 54. The separated sample components are detected by a detection beam 56, such as a beam of ultraviolet light, as they pass a detection point 55. Sphere 52
Inlet and outlet sections 58,5 for continuous flow of heat transfer fluid
Encased or submerged in a jacket or cooling vessel 57 with 9. Buffer solution reservoir 6 with electrodes 62, 63
0 and 61 are included as well as a sample reservoir 64 for sample filling, which can be replaced with one of the buffer solution reservoirs 61 once the sample is filled. In the figure,
The parts are arranged for sample filling. As an alternative to the arrangement shown, the left electrode 62 may be placed inside the sphere 52 rather than another reservoir.

In this embodiment, the parameter governing the amount of liquid drawn into the capillary by the temperature difference applied to the sphere is
It is the volume of the sphere, the temperature difference, and the coefficient of thermal expansion of the contents of the sphere. The contents of the sphere may be the same medium present in the separating portion 51 of the capillary or two different. It is most convenient to use spheres filled with the same separation medium as in the capillaries.
In the arrangement shown, liquid is drawn in from both sides of the sphere. However, in the above aspect, the system
It is adapted to draw liquid only through the capillary to the right side. The above examples apply here as well, namely the use of high clay liquid in the buffer reservoir 60 on the left or the use of a dialysis membrane or gel plug on the left side of the sphere. In any case, the sphere is
It must be filled with a thermally shrinkable medium.

Keeping these considerations in mind, the volumetric capacity of the sphere can vary widely without rigor. In many applications, the volume of the sphere will range from about 1 ml to about 100 ml.

The change in temperature is caused by changing the temperature of the heat transfer fluid flowing through the jacket 57 surrounding the sphere. The amount of change will, of course, be chosen according to the amount of liquid that is sought to be drawn into or through the separating portion 51 of the capillary. Therefore, a small temperature change is sufficient to load the sample into the capillary, while a large temperature change is used to flush the entire separated portion of the capillary with the buffer solution.

In another embodiment of the present invention, both temperature control spheres and temperature control capillaries are used simultaneously to provide flexibility to the various procedures that follow, and to provide effective temperature control of the capillaries at all times.

The principle embodied in the sphere aspect can be applied to various systems. An automated system designed for a series of separations carried out in succession is particularly advantageous according to the invention.

One example of this system is shown schematically in FIG. As in the system described in FIG. 3, electrodialysis separation occurs at the capillary 70, and bulk movement inside the capillary is controlled by the jacketed sphere 71.

On the opposite end of the capillary 70, a recess for sample and buffer
There is a carousel 72 including 73. In the front view of the carousel included in the figure, the depressions alternate with large depressions 74 for buffer and small depressions 75 for the sample. The surface of the carousel is a non-wettable material, such as Teflon, which allows the sample and buffer to form beads on the surface of the carousel when the depressions are flooded. When a very small sample is used, the carousel is placed in a laboratory hood or other chamber to keep the atmosphere saturated with water vapor to prevent the bead size from being reduced by evaporation.

The capillaries and carousel are positioned relative to each other such that the open ends 76 of the capillaries extend into the beads which are well coincident with the capillaries at all particular points. The electrodes 77, 78 are properly located, one 77 of which is associated with a buffer reservoir, but not necessarily in direct contact. The direction of migration of the sample during electrophoresis in this arrangement is to the right, as indicated by arrow 79, and detection is done online by the appropriate beam 80. Other arrangements of these elements that perform the same function will be readily apparent to those skilled in the art.

To prepare for operation, the capillary 70 and the bulb 71 are well filled with buffer using conventional means such as a syringe,
The carousel depressions are alternately filled with sample and buffer in an amount sufficient to form beads on the carousel surface. When the carousel is then rotated to bring the capillary into contact with the bead of sample and the stop valve 81 on the opposite side of the sphere is closed, a small temperature drop is applied to the sphere, reducing the volume of the small sample to the open end 76 of the capillary. To pull in.
While keeping the sphere at its lowered temperature, rotate the carousel to place a bead of buffer solution on one of the large buffer 74 depressions that are in contact with the ends of the capillary and apply an electrical potential across the electrodes. Perform electrophoresis.

Once the electrophoresis is complete, the capillary again prepares for the next sample by lowering the temperature of sphere 71,
The temperature is then sufficient to draw sufficient buffer through the capillary to flush it sufficiently. The carousel is then rotated into the next sample well and the whole process is repeated. The temperature of the sphere can be raised at any time without disturbing the contents of the capillaries by opening the outer stop valve 81 which releases fluid while closing the inner stop valve 82. As you can easily see, the capillary flush is
This can be accomplished by raising the temperature of the sphere and pushing the buffer solution through the capillary in the opposite direction.

The invention is applicable to a wide range of types of electrophoretic separations performed on capillaries. Examples of this type are simple electrophoresis, discontinuous electrophoresis, displacement electrophoresis and isoelectric focusing.

The following examples are provided by way of illustration and are in no way intended to define or limit the invention.

Example The device was assembled generally according to FIG. Capillaries are full length
It was a tube of fused silica with a length of 210 mm and a cooling length of 140 mm. The inner diameter of the tube was 0.05 mm. A buffer solution consisting of 0.1 M Tris acetic acid (pH 8.6) was used. The sample was prepared by dissolving 1 mg of 3,4-dimethoxybenzoic acid in 1 ml of a buffer solution and adding 1:10 with water.
It was a solution of 3,4-dimethoxybenzoic acid prepared by diluting.

Fill the capillary with buffer solution and let the tray at room temperature (20 ° C)
Was filled with water and the container of the right electrode was further filled with a buffer solution containing 0.1% methylcellulose. The container of the left electrode was filled with the sample solution. The water in the tray was then replaced with ice water (0 ° C.) and the sample rapidly drawn into the tube 0.5 mm long. This was consistent with the calculated length of 0.5 mm. The sample solution on the left electrode was then replaced with buffer and electrophoresis was started.

With the above tubes and solutions, the calculated length of the zone of packed sample and the length of the cooled part of the capillary for a given temperature drop are:

The foregoing has been provided primarily for purposes of explanation. It will be readily apparent to those skilled in the art that many changes, changes and substitutions can be made in the materials and methods disclosed herein without departing from the spirit and scope of the invention.

Claims (10)

[Claims] 1. A method for introducing a specific volume of liquid into a capillary in a capillary electrophoresis system, the capillary being filled with an electrophoretic separation medium, the method comprising: (a) one end of the capillary being in the body of the liquid. And (b) using the capillary so positioned to increase the temperature of a container filled with a fluid medium in fluid communication with the capillary by a preselected temperature difference. The fluid medium is varied in volume with temperature, thereby moving a portion of the liquid into the capillary, the magnitude of the temperature difference and the volume of the container being such that the amount of liquid so displaced. Equal to the specified volume. 2. The method of claim 1 wherein said container is a portion of said capillary and said fluid medium is a portion of said electrophoretic separation medium of said portion of said capillary. 3. The container is part of the capillary, step (b)
The method of claim 1 comprising lowering the temperature from about 10 to about 70 ° C. 4. The method of claim 1, wherein the container is a portion of the capillary that extends from about 50 to about 2000 mm in length, the capillary having an inner diameter of about 20 to about 100 microns. 5. The container is a portion of the capillary, the temperature difference and the length of the portion of the capillary being such that the specific volume is about 0.
The method of claim 1 selected to be between 0005 and about 0.005 μl. 6. The container is part of the capillary, the end of the opening of the capillary to the liquid in step (a) is defined as the inlet and the opposite end of the capillary is defined as the outlet. And the end of the outlet is submerged in an external liquid medium of a viscosity sufficiently higher than the separation medium for the duration of step (b) to allow said capillary to said capillary at said outlet due to said decrease in temperature. The method of claim 1, wherein any tendency to draw an external liquid medium is suppressed. 7. The electrophoretic separation medium is a liquid.
the method of. 8. A method of placing a particular volume of liquid sample inside the inlet and outlet ends and the inlet end of a capillary having an inner diameter of about 20 to about 100 microns for the purpose of electrophoretic separation. The capillary is filled with a separating liquid and the method comprises: (a) submerging the end of the inlet into the body of the liquid sample and the end of the outlet of a substantially higher viscosity exterior than both the liquid sample and the separation medium. And (b) passing the liquid heat exchange medium outside the central portion of the capillary, which has a length of about 100 to about 1000 mm, while (b) so submerging the inlet and outlet ends. The lowering of the preselected temperature lowers the temperature of the core, thereby contracting the liquid separation medium, causing a portion of the liquid sample to be drawn into the capillary at the end of the inlet, the preselected Temperature drop and the inside of the capillary The length of the core portion is selected such that the amount of liquid sample thus drawn into the capillary is equal to said specific volume. 9. The container is a sphere bound to the capillary, the volume of the sphere and the magnitude of the temperature difference being selected such that the specific volume is at least equal to the volume of the capillary. Method 1. 10. A method of separating a mixture of compounds in a liquid solution into components in a capillary having inlet and outlet ends and filled with a separation medium at a starting temperature, the method comprising: (a) Submerging the inlet end of the capillary into the body, and (b) lowering the temperature of at least a portion of the capillary below the starting temperature by a preselected temperature difference while so submerging the inlet end. Contracting the separation medium, thereby drawing a portion of the liquid solution into the end of the inlet of the capillary, (c) removing the end of the inlet of the capillary from the body of the liquid solution, and (D) A method comprising applying an electric potential between the capillaries and performing electrophoresis of the portion of the liquid solution thus drawn at the end of the inlet.